We are combining three technologies to obtain high resolution data on the distribution of antigens during the cell cycle or development: 1) optimized methods for fixation, using either cryofixation by high pressure freezing followed by freeze substitution or light aldehyde fixation followed by dehydration by progressive lowering of temperature; 2) embedding in methacrylate resins, and 3) 3-D reconstruction from serial sections for electron microscopy. We have worked out effective cryofixations for Drosophila and sea urchin embryos; adult and embryonic tissues of the nematode, C. elegans; mammalian tissue culture cells and extracellular matrix; and both fission and budding yeast cells. We have also worked out methods for flat-embedding these cells in LR White or Lowicryl resins. Most of our efforts so far have been directed at optimizing the quality of ultrastructural preservation and the density and specificity of immunolabeling. We have also developed image processing methods that identify colloidal gold particles on electron micrographs with about 99% accuracy. The resulting files of 2-dimensional coordinates can be used to compute gold distributions relative to any coordinate system. This technology is currently being applied to gamma tubulin, the tubulin characteristic of microtubule organizing centers. This form of tubulin is distributed around the centrosomes of mammalian mitotic cells, extending much farther from the centrioles than had previously been shown. We are now making models from serial sections, using existing programs for 3-D modeling, and date obtained from reconstructed MTs and 3-D coordinates of gold particles. We are also beginning work with semi-thick sections of cells which have been pre-embedded labeled with antibodies to gamma-tubulin with hope of obtaining a tomographic reconstruction of this antibody distribution.